Membrane biogenesis is essential for cell growth and differentiation. During membrane biogenesis in eukaryotic cells, newly synthesized phospholipids must be transported from their sites of synthesis to their sites of function. Vesicular traffic in eukaryotic cells is characterized by two steps of membrane rearrangement: the formation of vesicles from donor membranes and fusion of these vesicles with acceptor membranes. With respect to vesicle formation, several of the cytosolic proteins implicated in budding and fission have been identified. These stimulate the formation of constitutive secretory vesicles and immature secretory granules from the trans-Golgi network.
Phosphatidylinositol transfer protein (PITP) is a member of a diverse set of cytosolic lipid transfer proteins that are distinguished by their ability to transfer phospholipids between membranes in vitro and to take part in secretory vesicle formation (Wirtz, K. W. A. (1991) Ann. Rev. Biochem. 60:73-99; Ohashi, M. et al. (1995) Nature 377:544-547). PITP has been purified from mammals, plants, fungi and bacteria (Wirtz, K. W. A. (1991) supra).
PITPs have raised considerable interest because of their proposed roles in the phosphoinositide cycle and the ATP-dependent, Ca.sup.2+ -activated secretory process (Thomas, G. M. et al. (1993) Cell 74:919-928; Hay, J. C. and Martin, T. F. J. (1993) Nature 366:572-575). Furthermore, mammalian PITPs have a yeast counterpart, protein SEC 14p, which is an essential factor in the secretory vesicle flow from the trans-Golgi network to the plasma membrane (Bankaitis et al. (1989) J. Cell Biol. 108:1271-1281; Bankaitis et al. (1990) Nature 347:561-562) and shares no sequence homologies with the mammalian PITPs.
In mammals, two isoforms of PITP have been identified. Human testis PITP.alpha. has 78% identity with human brain PITP.beta. (Dickeson, S. K. et al. (1994) Gene 142:301-305; Tanaka S. et al. (1995) Biochim. Biophys. Acta 1259:199-202).
A PITP homologue has been identified in an insect retinal protein, Drosophila retinal degradation B (rdgB) protein (Vihtelic, T. S. et al. (1991) Genetics 127:761-768). Amino acid sequence identity is 40% between human PITP.alpha. and the 280 amino acid residue N-terminal domain of the fly protein. The N-terminal domain in fly rdgB protein has PI transfer activity an additional 803 residue C-terminal domain of rdgB is critical for proper protein function in flies (Vihtelic, T. S. et al. (1991) supra). Mutations present in the carboxy-terminal domain result in a truncated peptide and flies carrying the rdgB mutations undergo light-enhanced retinal degradation (Vihtelic et al. (1991) supra).
The yeast (Saccharomyces cerevisiae) PITP is essential for cell growth (budding) (Bankaitis et al. (1990) supra) and is presumed to be critical for membrane synthesis during budding, as well as being a component of the PI second messenger system that is associated with the cell cycle. In support of the latter role, it has been suggested that yeast PITP synergizes with a type I PI-4-phosphate 5-kinase to promote priming of dense-core secretory granules for Ca.sup.2+ -regulated fusion to the plasma membrane and that a significant aspect of the ATP requirement in vesicle trafficking may be dedicated to phosphatidylinositolbisphosphate (PIP.sub.2) synthesis (Hay, J. C. and Martin, T. F. J. (1995) supra).
Human PITP.alpha. is also required for the PI-phospholipase C-mediated hydrolysis of PIP.sub.2 in response to plasma membrane epidermal growth factor receptor stimulation (Kauffman-Zeh, A. et al. (1995) Science 268:1188-1190). Kinetic data argues strongly that PITP recruits PI from intracellular compartments, presents PI to PI 4-kinases in both the plasma membrane and the nucleus, and is a cofactor in inositol lipid signaling (Cunningham, E. et al. (1995) Curr. Biol. 5:775-783; Capitani, S. et al. (1991) Adv. Enzyme Reg. 31:399-416).
Mutation of rat PITP.alpha. at Thr59, which can be phosphorylated by protein kinase C (PK-C), regulates the binding affinity of PITP for PI, but not for phosphatidylcholine (PC) (Alb, J. G. et al. (1995) Proc. Natl. Acad. Sci. USA 92:8826-8830). Covalent modification of other residues within PITP has not yet been reported.
The etiology of numerous human diseases and disorders can be attributed to defects in the trafficking of proteins to organelles or the cell surface. Defects in the trafficking of membrane-bound receptors and ion channels are associated with cystic fibrosis, glucose-galactose malabsorption syndrome, hypercholesterolemia, and forms of diabetes mellitus. Abnormal hormonal secretion is linked to disorders including diabetes insipidus, hyper- and hypoglycemia, Grave's disease and goiter, and Cushing's and Addison's diseases.
The product of phospholipase C.beta.-mediated PI hydrolysis, inositol trisphosphate (InsP.sub.3), is associated with a number of cellular responses in diverse tissue types. These include glycogen breakdown in the liver, amylase and insulin secretion in the pancreas, smooth muscle contraction, histamine secretion from mast cells, and serotonin and platelet-derived growth factor secretion from blood platelets (McCance, K. L. and Heuther, S. (1994) Pathophvsiology (2nd Ed.) Mosby-Year Book, Inc. St. Louis, Mo. p. 20).
The critical role of InsP.sub.3 as a second messenger in the cell cycle suggests that decreasing the levels of membrane-associated PI within neoplastic tissue will contribute to the availability of PI as a source for InsP.sub.3 and prevent neoplastic growth stimulated by Ca.sup.2+ release from intracellular stores. In parallel, levels of intracellular diacylglycerol, the lipid product of PI hydrolysis, would be decreased and activation of the PK-C-mediated kinase cascade would be downregulated (Berridge, M. J. (1995) BioEssays 17:491-500).
The discovery of a new human phosphatidylinositol transfer protein and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention and treatment of disorders associated with abnormal vesicle trafficking and neoplastic disorders.